The presence of microRNAs (miRNAs) in both human and animal circulating blood has been reported (Chen, et al. (2008) Cell Res. 18:997-1006). Serum or plasma miRNAs may serve as novel clinical biomarkers for diverse diseases, because the levels of these cell-free serum miRNAs are significantly changed in human under disease conditions (Cortez & Calin (2009) Expert Opin. Biol. Ther. 9:703-711).
MicroRNAs are endogenous non-coding single-stranded RNAs of approximately 22 nucleotides in length and constitute a novel class of gene regulators (Chua, et al. (2009) Curr. Opin. Mol. Ther. 11:189-199). Analogous to the first RNA revolution in the 1980s (Zaug & Cech (1986) Science 231:470-475), the more recent discoveries of RNAi (RNA interference) and miRNA may represent the second RNA revolution. Although the first miRNA, lin-4, was discovered in 1993 (Lee, et al. (1993) Cell 75:843-854; Wightman, et al. (1993) Cell 75:855-862), their presence in vertebrates was only confirmed in 2001 (Lagos-Quintana, et al. (2001) Science 294:853-858). Currently, approximately 800 miRNAs have been cloned and sequenced in humans (Bentwich, et al. (2005) Nat. Genet. 37:766-770), and the estimated number of miRNA genes is as high as 1000 in the human genome (Lewis, et al. (2005) Cell 120:15-20).
Mature miRNAs bind to the 3′-UTR (untranslated region) of their mRNA targets and negatively regulate gene expression via degradation or translational inhibition (Chen & Rajewsky (2007) Nat. Rev. Genet. 8:93-103). Functionally, an individual miRNA is important as a transcription factor because it is able to regulate the expression of its multiple target genes. As a group, miRNAs are estimated to regulate over 30% of the genes in a cell. It is thus not surprising that miRNAs are involved in the regulation of almost all major cellular functions, including apoptosis and necrosis. Accordingly, miRNAs may be involved in many diseases, including cardiovascular disease (Zhang (2008) Clin. Sci. 114:699-706; Zhang (2008) Physiol. Genomics 33:139-147).
Tissue- and cell-specific expression is one important characteristic of miRNA expression (Lagos-Quintana, et al. (2002) Curr. Biol. 12:735-739). Indeed, one miRNA may be highly expressed in one tissue or one cell, but has no or low expression in other tissues or cells. For example, miR-1 is reported to be a muscle or heart-specific miRNA, whereas miR-145 is a vascular smooth muscle cell-specific miRNA (Cheng, et al. (2009) Circ. Res. 105:158-166). The tissue-specific miRNA expression and tissue expression signatures of diseases have provided a great diagnostic opportunity for diverse diseases (Dong, et al. (2009) J. Biol. Chem. 284:29514-29525).
Recent studies have revealed that miRNAs exist in circulating blood (Ji, et al. (2007) Circ. Res. 100:1579-1588). Cell-free miRNAs are relatively stable due to binding with other materials such as exosomes in circulating blood. Moreover, cancer tissue miRNAs are able to be released into circulating blood and serum or plasma cell-free miRNAs can be used as novel biomarkers for diverse cancers. However, a robust quantitative method to measure the absolute amount of a miRNA in blood has not been established due to a lack of stable control RNAs in blood, especially under disease conditions. More importantly, the role of the circulating cell-free miRNAs in patients with cardiovascular diseases is currently unclear. Therefore, there is a need in the art for improved assays for isolating and analyzing miRNAs in biological fluids.